Diagnosis and treatment of human kidney diseases

ABSTRACT

Kidney disease is diagnosed by measuring urinary catalytic iron in humans. Progressive kidney disease is treated by administering an iron chelator to humans. In particular, the progression of kidney disease essentially can be halted and the severity of kidney disease can be reduced by the administration of iron chelators to humans afflicted with a progressive kidney disease. The methods include measuring catalytic iron content in urine in a human afflicted with a progressive kidney disease and administering an iron chelator to the human. The method can include measuring total urinary protein content, blood urea nitrogen or creatinine in a blood sample before, during or after the administration of an iron chelator.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/553,496filed Apr. 20, 2000 which claims the benefit of U.S. ProvisionalApplications Ser. Nos. 60/130,903 and 60/130,908, both filed Apr. 23,1999, the entire teachings of both of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Kidney disease can be progressive, leading to end-stage renal diseaseand, ultimately, death. End-stage renal disease affects approximately650,000 human patients each year worldwide and treatment costs have beenestimated to be about twenty billion dollars (Klahr, S., Contrib.Nephrol. 118:1-5 (1996)).

Current methods to diagnose kidney disease and, in particular,progressive kidney disease, include monitoring urine for elevatedprotein levels and conducting renal biopsies. Available treatments forkidney disease, specifically progressive kidney disease, include the useof steroids, alkylating agents and cyclosporine (de Mattos, A. M. etal., pages 861-885, In: “Immunologic Renal Diseases,” eds. Neilson, E.G. et al., Lippincott-Raven Publishers, Philadelphia, Pa. (1997)). Theabove mentioned diagnostic methods often are inadequate sincesignificant damage to the kidney can occur prior to diagnosis. Likewise,the above mentioned treatments frequently are unsuccessful in haltingthe progression of kidney disease and, therefore, unsatisfactory, sincethey often are accompanied by adverse side effects, such as cellular andsystemic toxicity.

Although animal models have been used to study some kidney diseases,animal models suffer from a number of limitations when extended to humankidney disease diagnosis and treatment. For example, animal models ofkidney disease do not necessarily depict kidney disease in humans andthe administration of steroids, although reasonably effective in thetreatment of kidney disease in experimental animal models, arerelatively ineffective in the treatment of kidney disease in humans(Siegel, N. J. et al., Kidney Int. 25:906-911 (1984); Cronin, R. E., etal., Am. J. Physiol. 248:F332-F339 (1985); Cronin, R. E., et al., Am. J.Physiol. 251:F408-F416 (1986); Seiken, G. et al., Kidney Int.45:1622-1627 (1994); Shaw, S. G. etal., J. Clin. Invest. 80:1232-1237(1987); Allgren, R. L. et al., N. Eng. J. Med. 336:828-834 (1997);Paller, M. S., Sem. Nephrol. 18:482-489 (1998); Savill, J., et al., In:“Oxford Textbook of Clinical Nephrology”, eds. Davison, A. M., et al.,Oxford Medical Publications, page 403-439 (1998); Hirschberg, R. et al.,Kidney Int. 55:2423-2432 (1999)). Thus, animal model systems generallycan not be used to define and identify methods which halt theprogression, reduce the severity, treat, or diagnose progressive kidneydisease in humans. Thus, there is a need to develop new, improved andeffective methods of reducing the severity, diagnosing and treatingkidney disease in humans.

SUMMARY OF THE INVENTION

The present invention relates to methods of diagnosing and treatingkidney disease in a human. In particular, the invention relates tomethods of treating progressive kidney diseases.

In one embodiment, the method includes diagnosing a kidney disease in ahuman comprising the step of measuring catalytic iron in a urine sample.In a preferred embodiment, the catalytic iron is measured in relation toa reference protein. In a particularly preferred embodiment, thereference protein is urinary creatinine.

In another embodiment, the invention relates to a method of treating akidney disease in a human comprising measuring catalytic iron content ina urine sample obtained from the human and comparing catalytic ironcontent in the urine sample with catalytic iron content in a controlsample. A catalytic iron content in the urine sample above catalyticiron content in the control sample is indicative of kidney disease.

In yet another embodiment, the invention includes a method of treating akidney disease in a human, wherein the human has a catalytic ironcontent in urine greater than about 15 nmol/mg of a reference proteincomprising administering an iron chelator to the human.

In an additional embodiment, the invention includes a method of treatinga kidney disease in a human comprising administering a dose of an ironchelator to a human having catalytic iron content in urine exceedingthat of a control sample and obtaining a first urine sample from thehuman. The catalytic iron content in the first urine sample is measuredand compared to the catalytic iron content in the catalytic iron contentin the control sample.

The method can further include administering at least one subsequentdose of the iron chelator. The amount of the subsequent dose isdetermined by comparison of the measured catalytic iron content in theurine sample to the catalytic iron content in the control sample.

In another embodiment, the method further includes obtaining at leastone subsequent urine sample and measuring catalytic iron content in thesubsequent urine sample. The catalytic iron content in the subsequenturine sample is compared to the catalytic iron content in the firsturine sample, the control sample or both.

Yet another embodiment of the invention includes determining progressionof kidney disease in a human. Catalytic iron content in a first urinesample and at least one additional urine sample obtained from the humanis measured. Catalytic iron content in the additional urine sample iscompared with catalytic iron content in the first urine sample. Anelevation in catalytic iron content in an additional urine sample abovecatalytic iron content in the first urine sample is indicative of theprogression of the kidney disease in the human.

In still another embodiment, the invention relates to a method ofevaluating the effectiveness of administering an iron chelator to treata human suffering from a progressive kidney disease. Catalytic ironcontent in a first urine sample obtained from the human is compared withcatalytic iron content of a subsequent urine sample obtained from thehuman. The subsequent urine sample is obtained after obtaining the firsturine sample and after the administration of the iron chelator. Acomparison of the catalytic iron content in the subsequent urine sampleand the first urine sample is indicative of the effectiveness of theadministration of the iron chelator to alleviate progression of thekidney disease.

In another embodiment, the invention relates to a method of identifyinga human suffering from a progressive kidney disease who will benefitfrom treatment of the progressive kidney disease with the iron chelator.Catalytic iron content in a urine sample obtained from the human ismeasured. A catalytic iron content in the urine sample greater thanabout 15 nmol/mg of a reference protein identifies a human sufferingfrom a progressive kidney disease who will benefit from treatment of theprogressive kidney disease with the iron chelator.

In yet another embodiment, the invention relates to a method of treatingmicroalbuminuria in a human comprising administering a dose of an ironchelator to a human suffering from microalbuminuria having a catalyticiron content in urine exceeding a control sample. A first urine sampleis obtained from the human and catalytic iron content measured in thefirst urine sample. The catalytic iron content in the first urine sampleis compared with the catalytic iron content in the control sample.

The invention described herein provides methods for diagnosing,treating, halting the progression and reducing the severity of kidneydisease in a human. Advantages of the claimed invention include, forexample, treatment of progressive kidney disease in a human in a mannerwhich has not been described in an animal model, nor could not bepredicted from existing animal models of kidney disease. The methods ofthe invention can treat progressive kidney diseases in humans withoutsignificant side effects. The methods of the invention provide aneffective manner to treat kidney disease and, ultimately, preventend-stage renal disease.

Thus, treatment of humans who have a catalytic iron content in the urineabove a control level with iron chelators, can halt, reverse, or reducethe severity of the progression of kidney disease, thereby increasingquality of life and life expectancy.

BRIEF DESCRIPTION OF THE FIGURES

The FIGURE represents the catalytic iron content (nmol/mg urinarycreatinine) in urine obtained from humans with no kidney disease(control), with diabetic microalbuminuria (DM Micro), diabetic (DM)proteinuria, glomerulonephritis (GN) and ischemic nephrophathy.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of theinvention or as combinations of parts of the invention, will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

The present invention relates to the discovery that catalytic iron inurine of a human can be an indicator of the existence of kidney disease.The invention further relates to the discovery that the administrationof an iron chelator decreases protein content in urine of humanssuffering from kidney diseases. In particular, the iron chelatordeferiprone has been found to decrease protein content and serumcreatinine, both indices of kidney damage, in urine obtained from humanssuffering from progressive kidney disease.

“Kidney disease” generally refers to a disorder of at least one kidneyin a human that compromises the function of the kidney (e.g., to preventleakage of protein, to prevent excretion of nitrogenous waste). Thekidney disease can result from a primary pathology of the kidney (e.g.,injury to the glomerulus or tubule) or another organ (e.g., pancreas)which adversely affects the ability of the kidney to perform biologicalfunctions (e.g., retain protein). Thus, kidney disease in the human canbe the direct or indirect effect of disease. Examples of a kidneydisease as a result or consequence of an indirect effect on the kidneysis kidney disease as a consequence of diabetes or systemic lupus. Theterm kidney disease is used interchangeably with the phrase “diseases ofthe kidney.” The kidney disease can be, for example, a result or aconsequence of any change, damage, or trauma to the glomerulus, tubulesor interstitial tissue in either the renal cortex or renal medulla ofthe kidney.

In a preferred embodiment of the invention, the kidney disease is aprogressive kidney disease. “Progressive kidney disease” as used hereinrefers to any disease of the kidney that over time (e.g., days, weeks,months, years) leads to a loss of renal function. “Renal function”generally refers to a physiological property of the kidney, such as theability to retain protein thereby preventing proteinuria (e.g., urinarycreatinine, the excretion of protein in an amount greater than about0.15 g/24 hours). Renal function can be assessed, for example, byglomerular filtration rate (e.g., creatinine clearance), excretion ofprotein in urine, blood urea nitrogen, serum or plasma creatinine, orany combination thereof.

Glomerular filtration rate is an indicia of renal function whichgenerally refers to the renal (also referred to herein as kidney)excretory capacity. Indices to assess glomerular filtration include, forexample, creatinine clearance and inulin clearance. Exemplarydescriptions and discussions of techniques to assess renal function,including glomerular filtration rate (e.g., creatinine clearance) arefound, for example, in Larsen, K. Clin. Chem. Acta. 41:209-217 (1972);Talke, H. et al., Klin, Wscr 41:174 (1965); Fujita, Y. et al., BunsekiKgaku 32:379-386 (1983); Rolin, H. A. III, et al., In: “The Principlesand Practice of Nephrology”, 2nd ed., Jacobson, H. R., et al.,Mosby-Year Book, Inc., St. Louis, Mo., page 8-13 (1995); Carlson, J. A.,et al., In: “Diseases of the Kidney”, 5th Ed., Schrier, R. W. et al.,eds., Little, Brown and Co., Inc., Boston, Mass., pages 361-405 (1993),the teachings of all of which are hereby incorporated by reference intheir entirety. For example, endogenous creatinine clearance can bedetermined as follows:Cr=Ucr V/PCr

-   -   where Cr=clearance of creatinine (ml/min); Ucr=urine creatinine        (mg/dl), V=volume of urine (ml/min−for 24-hr volume: divide by        1400, and PCr=plasma creatinine (mg/dl).

A progressive kidney disease treated by the methods described hereinincludes any kidney disease that can, ultimately, lead to end-stagerenal disease. A progressive kidney disease of the invention is not adisease of the kidney that results from exogenous iron overload as aresult of, for example, repeated blood transfusions in diseases such asthalassemia or sickle cell anemia. The progressive kidney diseases thatcan be diagnosed or treated by the methods of the invention can be, forexample, associated with endogenous iron deposit in the kidney (e.g.,glomerulus, tubules). The endogenous iron can be released, for example,from ferritin, mitochondria or cytochrome P450 in the human.

In a more preferred embodiment, the kidney disease is a progressiveglomerular kidney disease. Progressive glomerular kidney diseases thatare particularly suitable for treatment by the method of the inventioninclude, for example, diabetic nephropathy (e.g., as a consequence ofType I or Type II diabetes or systemic lupus), primaryglomerulonephritis (e.g., membranous nephropathy, focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, diffuseproliferative glomerulonephritis, membranous focal segmentalglomerulosclerosis) and secondary glomerulonephritis (e.g., diabeticnephropathy, ischemic nephropathy).

A kidney disease can be diagnosed in a human by the method of theinvention by measuring the catalytic iron in urine of the human. A humanis also referred to herein as a patient or an individual. In a preferredembodiment, a human can be diagnosed with a kidney disease whencatalytic iron content in urine is greater than about 15 nmol/mg of areference protein.

“Catalytic iron” refers to Fe²⁺. Catalytic iron is capable of catalyzingfree radical reactions. Iron in the Fe²⁺ state can catalyze theHaber-Weiss reaction, which reduces hydrogen peroxide and promotesformation of hydroxyl radicals. The Haber-Weiss reaction is illustratedbelow: $\begin{matrix}\begin{matrix}\left. {O_{2}^{-} + {Fe}^{3 +}}\rightarrow{O_{2} + {Fe}^{2 +}} \right. \\\left. {{Fe}^{2 +} + {H_{2}O_{2}}}\rightarrow{{Fe}^{3 +} + {{OH}\bullet} + {OH}^{-}} \right.\end{matrix} \\\left. {O_{2}^{-} + {H_{2}O_{2}}}\rightarrow{O_{2} + {{OH}\bullet} + {OH}^{-}} \right.\end{matrix}\quad$

It is also envisioned that catalytic iron can result in the formation ofhydroxyl radicals in reactions other than the Haber-Weiss reaction.

Methods to determine the catalytic iron content are known (see, forexample, Gutteridge, J. M. C. et al., Biochem J. 199:263-265 (1981);Yergey, A. L. J. Nutrition 126:355S-361S (1996); Iancu, T. C. et al.,Biometals 9:57-65 (1996); Artiss, J. D. et al., Clin. Biochem.14:311-315 (1981); Smith, F. E. et al., Clin. Biochem. 17:306-310(1984); Artiss, J. D., et al., Microchem. J. 28:275-284 (1983), theteachings of all of which are hereby incorporated by reference in theirentirety) and as described in the exemplification. The methods includespectrophotometric, mass spectrometry (e.g., thermal ionization massspectrometry, laser microprobe mass analysis, inductively coupled plasmamass spectrometry, atom bombardment-secondary ion mass spectrometry).Catalytic iron content is also referred to as an amount, level orconcentration of catalytic iron.

In a preferred embodiment, the catalytic iron content is measured inrelation to a reference protein in the urine sample. The phrase “inrelation to a reference protein” as used herein refers to expressingcatalytic iron content in urine as a unit of measurement (e.g.,nanomoles) per a concentration of a protein (e.g., milligrams) in theurine. Catalytic iron can be expressed, for example, as nanomoles(nmoles) of catalytic iron per milligram (mg) of protein in urine. In apreferred embodiment, the reference protein is urinary creatinine.

Additionally, or alternatively, catalytic iron content is measured inrelation to glomerular filtration rate, in particular creatinineclearance as described above.

Preferably, catalytic iron content is measured in a urine sample from ahuman prior to the onset of clinical symptoms (also referred to hereinas clinical signs of kidney disease) of kidney disease (e.g.,proteinuria, increase blood urea nitrogen, increase serum creatinine).Additionally, or alternatively, catalytic iron content is measured in aurine sample obtained from the human after the onset of clinicalsymptoms of kidney disease (e.g., proteinuria). Catalytic iron ismeasured in humans afflicted with a kidney disease and compared tohumans not afflicted with a kidney disease (See Tables 1 and 2).

An “iron chelator” refers to any molecule capable of interacting withiron, either Fe³⁺ or Fe²⁺, to prevent the formation of catalytic ironfrom Fe³⁺ or to prevent, inhibit or interfere with iron (Fe³⁺ or Fe²⁺)interacting, effecting or participating in the Haber-Weiss reaction orany other reaction which can generate hydroxyl radicals. The interactionbetween the iron chelator and iron, either Fe³⁺, Fe²⁺, or both, can be,for example, a binding interaction, an interaction as a result of sterichindrance or any reciprocal effect between iron and the iron chelator.The iron chelator can, for example, prevent the conversion of Fe³⁺ toFe²⁺, thereby indirectly preventing the reduction of hydrogen peroxideand formation of hydroxyl radicals in the Haber-Weiss reaction.Alternatively, or additionally, the iron chelator can interact directlywith Fe²⁺ to prevent hydroxyl radical formation in, for example, theHaber-Weiss reaction.

The iron chelator can be a peptide comprising natural or nonnatural(e.g., amino acids not found in nature) amino acids, polyethylene glycolcarbamates, lipophilic or nonlipophilic polyaminocarboxylic acids,polyanionic amines or substituted polyaza compounds. In a preferredembodiment, the iron chelator is deferiprone(1,2-dimethyl-3-hydroxy-pyrid-4-one)L1. Iron chelators are commerciallyavailable or can be synthesized or purified from biological sourcesusing routine procedures. Exemplary descriptions and discussions of ironchelators are found in several references, for example, U.S. Pat. No:5,047,421 (1991); U.S. Pat. No: 5,424,057 (1995); U.S. Pat. No:5,721,209 (1998); U.S. Pat. No: 5,811,127 (1998); Olivieri, N. F. etal., New Eng. J. Med. 332:918-922 (1995); Boyce, N. W. et al., KidneyInternational. 30:813-817 (1986); Kontoghiorghes, G. J. Indian J.Peditr. 60:485-507 (1993); Hershko, C. et al., Brit. J. Haematology101:399-406 (1998); Lowther, N. et al., Pharmac. Res. 16:434 (1999), theteachings of all of which are hereby incorporated by reference in theirentirety.

An “amount effective,” when referring to the amount of iron chelator isdefined as that amount (also referred to herein as dose) of ironchelator that, when administered to a human afflicted with a kidneydisease, is sufficient for therapeutic efficacy (e.g., an amountsufficient to reduce the catalytic iron content in urine, total proteincontent in urine, blood urea nitrogen, or creatinine in a blood sampleobtained from a human with a progressive kidney disease). An effectiveamount of an iron chelator also refers to an amount of iron chelatorthat when administered to the human prevents a further or additionalincrease in urinary catalytic iron content compared to the catalyticiron content measured in urine prior to treatment or during treatmentwith the iron chelator. Catalytic iron can be measured in urine ofhumans undergoing treatment with iron chelator before, during or afterthe administration of the iron chelator.

In one embodiment, the iron chelator is administered in a single dose.In another embodiment, the iron chelator is administered in multipledoses. In a preferred embodiment, the iron chelator is administeredorally at a dose in a range between about 20 mg/kg body of the human andabout 150 mg/kg body weight of the human. In a particularly preferredembodiment, the iron chelator is administered three times a day at adose in a range of between about 20 mg/kg body of the human and about150 mg/kg body weight of the human for at least 2-6 months.

Another aspect of the invention relates to measuring protein content inurine of a human afflicted with a progressive kidney disease undergoingtreatment of the kidney disease with an iron chelator. A measure ofprotein content can be total protein in the urine (also referred toherein as total protein content). Protein content in urine of the humanafflicted with the kidney disease can be measured at one or more timepoints before, during or after the administration of the iron chelator.

Typically, protein content in urine is determined using routine assayswell-known to one of skill in the art. Suitable methods includedipstick, immunoprecipitation, turbidimetric (e.g., sulphosaliclic acid,tricholoracetic, benzethonium chloride) assays, dye-binding (e.g.,Coomassie Blue, Ponceau) assays, Biuret (e.g., precipitation withTsuchiya reagent) assays and Folin-Lowry assays. (“Oxford Textbook ofClinical Nephrology” eds. Davison, A. M., et al., 2nd edition, OxfordUniversity Press, New York, N.Y. (1998); “Primer on Kidney Diseases”ed., Greenberg, A., 2nd edition, Academic Press, New York, N.Y. (1998),the teachings of all of which are hereby incorporated by reference intheir entirety).

In another embodiment, creatinine, blood urea nitrogen, or both aremeasured in a blood sample obtained from the human at one or more timepoints before, during or after the administration of the iron chelatorto a human afflicted with a kidney disease. The blood sample can be anarterial blood sample or venous blood sample. The blood sample can be aserum or plasma blood sample. Methods to obtain blood samples andprocess the blood sample to determine blood urea nitrogen content andcreatinine content are well known to one of skill in the art. (See, forexample, Karlinsky, M. L. et al., Kidney Int. 17:293-302 (1980);Tomford, R. C. et al., J. Clin. Invest. 68:655-664 (1981); Baliga, R. etal., Biochem J. 291:901-905 (1993), the teachings of all of which areincorporated herein by reference in their entirety).

Blood urea nitrogen, urinary protein content or serum creatinine contentin a human afflicted with a kidney disease can increase in the human asthe kidney disease progresses. Likewise, blood urea nitrogen, totalurinary protein content and serum creatinine content in a humanafflicted with a kidney disease can decrease or stabilize (e.g., remainthe same, essentially constant) when the progression of a kidney diseaseis halted or prevented by the administration of iron chelators using themethods of the invention.

In a preferred embodiment, the iron chelator is administered to thehuman with a kidney disease in an amount effective to lower totalprotein content in urine, blood urea nitrogen or creatinine content in ablood sample of the human to about that of a control level.

In another embodiment, the iron chelator is administered to the humanwith a kidney disease in an amount effective to prevent any furtherincrease in total protein content in urine, blood urea nitrogen andcreatinine content in a blood sample of the human compared to an amountof catalytic iron prior to treatment with the iron chelator (alsoreferred to herein as a pretreatment amount of urinary catalytic iron).

In yet another embodiment, the iron chelator is administered to thehuman with a kidney disease in an amount effective to prevent anincrease in total protein content in urine, blood urea nitrogen andcreatinine in a blood sample of the human to a level above a controllevel.

In particular, the invention relates to a method of treating a kidneydisease in a human, comprising administering a dose of an iron chelator(e.g., in a range between about 20 mg iron chelator/kg body weight andabout 150 mg iron chelator/kg body weight) to a human having a catalyticiron content in urine exceeding that of a catalytic iron content in acontrol sample (e.g., above about 15 nmol catalytic iron/mg of areference protein). The dose which the human is initially treated withis also referred to as an “initial dose.”

In one embodiment, a urine sample is obtained from a human before theadministration of the iron chelator (also referred to herein as apretreatment urine sample). In another embodiment, a urine sample isobtained after the administration of the iron chelator. Thus, a urinesample can be obtained from the human before, during or after the humanis administered the iron chelator and catalytic iron content measured.

In a preferred embodiment, the iron chelator (e.g., deferiprone) isadministered to a human with a catalytic iron content in a urine samplein an amount greater than about 15 nmol/mg of a reference protein (e.g.,urinary creatinine) in a dose in a range of between about 20 mg/kg bodyweight and about 150 mg/kg body weight of the human.

As defined herein, “control sample” means a level (also referred toherein as amount or content) of the parameter of interest (e.g.,catalytic iron content in urine, protein content in urine, blood ureanitrogen in a blood sample, creatinine in a blood sample) in a human notafflicted with a kidney disease, matched, as necessary, for variables,such as age, sex, ethnicity and health history, with the human to betreated. A control sample can also be the expected level of theparameter of interest in a human. The “expected level” of the parameterof interest in a human treated by the methods of the invention can be alevel normally observed in a human not afflicted with the kidneydisease, or can be higher than a human not afflicted with the kidneydisease yet below the level in a human with a kidney disease. Anexpected level can also be any level of the parameter of interest thatis below pretreatment levels or lower than a level expected duringprogression of a kidney disease in a human not undergoing treatment bythe methods defined herein. A “target level” of the parameter ofinterest can be selected for a human afflicted with a kidney diseasebased on a level of the parameter of interest (e.g., catalytic ironcontent in the urine) of a human before that human developed a kidneydisease, or in comparison to levels observed in a human not exhibiting akidney disease. In a preferred embodiment, a control sample has acatalytic iron content of about 15 nmol/mg of a reference protein (e.g.,creatinine).

After administration of the iron chelator, the catalytic iron content inthe human is measured in a first urine sample. A “first urine sample,”when referring to methods of treating the progressive kidney disease,refers to a urine sample obtained from the human at any time after theadministration of the iron chelator. The first urine sample can beobtained, for example, days, weeks, months, or years after theadministration of the iron chelator. The catalytic iron content in aurine sample (e.g., first urine sample) is compared to the catalyticiron content in the control sample. The catalytic iron content in thefirst urine sample can also be compared to the catalytic iron content ina urine sample obtained from the human prior to or after administeringthe initial dose of the iron chelator.

The method can further include administering a subsequent dose of theiron chelator to the human. The amount of the subsequent doseadministered is determined by comparing the catalytic iron in the firsturine sample with the catalytic iron content in the control sample.

The method can further include obtaining as least one subsequent urinesample from the human. A “subsequent urine sample” is any urine sampleobtained from the human at any time after the first urine sample. One ormore subsequent urine samples can be obtained from the human. More thanone subsequent urine sample can be obtained in a day or over severaldays, months or years.

The catalytic iron content in the subsequent urine sample of the humanis compared with the catalytic iron content in the first urine sample,the control sample, or both. The dose of the iron chelator administeredto the human can be adjusted (e.g., increased) until the catalytic ironcontent in the urine of the human is about that of the control sample oruntil the catalytic iron content in the subsequent urine sample isessentially constant. The adjusted dose is also referred to as a“subsequent dose.” For example, if the human was administered about 75mg/kg body weight dose of iron chelator and the catalytic iron contentin a subsequent urine sample was about 35 nmol/mg of a reference proteincompared to about 55 nmol/mg of a reference sample in the urine sampleor first urine sample, the subsequent dose of iron chelator could beadjusted to below about 75 mg/kg body weight.

In another embodiment of the invention, the subsequent dose of the ironchelator is administered until the catalytic iron in a subsequent urinesample is about that of the control or essentially constant.“Essentially constant” (also referred to herein as “an essentiallyconstant value” or stabilized) refers to a catalytic iron content in theurine that remains about the same value over time (e.g., days, weeks,months, or years).

In a preferred embodiment, when the catalytic iron content in urine isabout that of a control sample or essentially constant, the dose of ironchelator in at least two subsequent doses differs by about 10 mg/kg ofbody weight of the human. After a 10 mg/kg increment adjustment, thecatalytic iron content in one or more subsequent urine samples can bemeasured, compared to the control or an essentially constant value andthe dose of iron chelator adjusted (e.g., increased) if the catalyticiron content in the subsequent urine sample is above a control sample orthe essentially constant value. For example, if the catalytic ironcontent in a subsequent urine sample of a human on a maintenance dose ofthe iron chelator is greater than the control sample (e.g., about 15nmol/mg of a reference protein) the dose of iron chelator can beincreased to decrease the catalytic iron in the urine of the humanafflicted with a kidney disease. Similarly, the dose of iron chelatorcan be decreased if the catalytic iron content in the subsequent urinesample remains, for example, at a control content or essentiallyconstant.

In yet another embodiment, urinary protein content (e.g., total proteincontent) in a urine sample (e.g., before or after treatment with theiron chelator) and at least one subsequent urine sample is measured. Theurinary protein content in the subsequent urine sample is determined andcompared to the protein content in a urine sample obtained before orafter iron chelator treatment, a control sample or both. Urinary proteincontent in a subsequent urine sample can be compared to a control sampleto determine the progress of treatment, the need for further treatment(e.g., number or duration of doses) or the need to adjust the dose(e.g., mg/kg body weight) of iron chelator being administered to thehuman.

“Adjusting the dose” of the iron chelator (also referred to herein as asubsequent dose of iron chelator) refers to any change or alteration(e.g., increase or decrease) in the amount of iron chelator in theinitial dose. A change in the amount of iron chelator can be, forexample, an increase or decrease in the frequency (e.g., times per day,number of days, number of months, number of years) of administration ofthe iron chelator. Additionally, or alternatively, a change in theamount of iron chelator can be an increase or decrease in the dose(e.g., milligrams of iron chelator per kilogram body weight of thehuman) of iron chelator administered to the human.

In another embodiment of the invention, creatinine content or blood ureanitrogen content in a blood sample obtained from the human is measuredat one or more points before, during or after administration of the ironchelator to a human afflicted with a kidney disease, and who has acatalytic iron content in urine greater than a control sample.

Additionally indices of reducing the severity of the progressive kidneydisease can be a decrease in the expected rate at which, for example,blood urea nitrogen, serum creatinine increases, glomerular filtrationrate declines, or the onset of end-stage renal disease is delayed. Adecline in glomerular filtration rate can be assessed, for example, byan increase in total urinary protein.

A blood sample obtained from the human before administration of an ironchelator is referred to as a “pretreatment blood sample.” A blood sampleobtained from the human after or during the administration of the ironchelator is also referred to as a “subsequent blood sample.” The bloodurea nitrogen content and creatinine content in at least one subsequentblood sample is compared with the blood urea nitrogen content andcreatinine content in at least one pretreatment blood sample or acontrol sample. Similarly, the blood urea nitrogen content andcreatinine content is compared in at least one subsequent blood sampleobtained from the human at different times following the administrationand during the course of iron chelator treatment.

The dose of iron chelator can be adjusted depending upon protein contentin urine, blood urea nitrogen, creatinine content in the subsequentblood sample, or any combination thereof. For example, a decline (alsoreferred to herein as a decrease) in urinary protein, blood ureanitrogen or creatinine content in a subsequent urine or blood samplecompared to a pretreatment sample or a control sample is indicative thatthe kidney disease is being treated by the iron chelator and the dose ofiron chelator could be decreased.

In another embodiment of the invention, a kidney disease in a human isdiagnosed. Catalytic iron content is measured in a urine sample obtainedfrom the human and compared with catalytic iron content in a controlsample. In this embodiment, catalytic iron content in the urine sampleobtained from the human above the catalytic iron content in the controlsample is indicative of kidney disease.

In another embodiment, the invention relates to a method of reducing theseverity of a progressive kidney disease in a human having a catalyticiron content in a urine sample greater than about 15 nmol/mg of areference protein. The human afflicted with the progressive kidneydisease (e.g., diabetic nephropathy, primary glomerulonephritis,secondary glomerulonephritis) is administered an iron chelator. The ironchelator decreases catalytic iron content in urine obtained from thehuman thereby reducing the severity of the progressive kidney disease.

The phrase “reducing the severity” (also referred to herein as areduction in the severity) when referring to a progressive kidneydisease means any diminution, amelioration or decrease in progressivedamage to the kidney that compromises the function of the kidney. Well-recognized indices to assess function of the kidney can be employed todetermine a reduction in the severity of the kidney disease. Theseindices can include, for example, a reduction in protein content inurine, a reduction in blood urea nitrogen, a reduction in serum orplasma creatinine, an increase in glomerular filtration rate, a delay inthe onset of end-stage renal disease, or any combination thereof,compared to a sample obtained from the human prior to administering theiron chelator, or a control sample.

A reduction in the severity of the kidney disease can result, forexample, from an amelioration of a primary pathology of the kidney(e.g., injury to the glomerulus or tubule) or another organ (e.g.,pancreas) which had adversely affected the ability of the kidney toperform biological functions (e.g., retain protein). Thus, a reductionin the severity of the kidney disease in the human can be the direct orindirect effect of a reduction of the kidney disease.

Another embodiment of the invention is a method of determining theprogression of a kidney disease in a human comprising measuringcatalytic iron in one or more urine samples obtained from the human. Anincrease in the catalytic iron content in a urine sample from the humanover time (e.g., weeks, months, years) is indicative of the progressionof the kidney disease. The catalytic iron content in urine can bemeasured relative to a reference protein (e.g., creatinine).

“Progression of a kidney disease” refers to an augmentation or increasein the disease of the kidney. Progression of a kidney disease caninclude, for example, continued or additional damage to a segment of thekidney (e.g., nephron, tubule, interstitium) that was not damaged whenthe human was initially afflicted or diagnosed with the kidney disease.For example, the human could have no glomerular damage or damage only tothe glomerulus resulting in a kidney disease which, over time, leads toincreased damage only to the glomerulus or results in tubular damage asthe kidney disease progresses. As the kidney disease progresses in thehuman, the catalytic iron content in urine can increase above a levelprior to additional damage to the kidney or in comparison to a human notaffected with a progressive kidney disease. Catalytic iron content inurine obtained from the human with progressive kidney disease can bedetermined and compared to urinary catalytic iron content in another,different human with various stages of progressive kidney disease todiscern the progression of the kidney disease.

Progression of the kidney disease can also be monitored, for example, bymeasuring protein content, in addition to catalytic iron content, inurine of the human afflicted with the kidney disease. As the kidneydisease progresses, the protein content in the urine can increase.Additionally, progression of the kidney disease in the human can beassessed by measuring the blood urea nitrogen content or creatininecontent in a blood sample obtained from the human over time. An increasein the blood urea nitrogen content or creatinine content over time inthe human is also indicative of the progression of the kidney disease inthe human. Similarly, a decline in glomerular filtration as assessed,for example, by creatinine clearance as described above can also be usedin conjunction with urinary catalytic iron content as indicative ofprogression of the kidney disease.

In another embodiment, the invention relates to a method of determiningthe progression of kidney disease in a human comprising measuringcatalytic iron content in a first urine sample and in at least oneadditional urine sample obtained from the human. When determining theprogression of a kidney disease, a “first urine sample” refers to aninitial sample of urine obtained from the human at anytime prior todetermining the progression of the kidney disease. An “additional urinesample” refers to a urine sample obtained from the human at any timeafter the first urine sample.

The catalytic iron content in the additional urine sample is compared tothe catalytic iron content in at least one additional urine sample. Anelevation in the catalytic iron in an additional urine sample comparedto a first urine sample is indicative of progression of the kidneydisease. Likewise, a reduction in the catalytic iron in an additionalurine sample compared to the first urine sample is indicative that thekidney disease is not progressing or is being reduced in severity.

The method of determining the progression of kidney disease in a humancan further include measuring protein content in the first urine sampleand at least one additional urine sample and creatinine content in afirst blood sample and at least one additional blood sample obtainedfrom the human afflicted with the kidney disease. A “first blood sample”refers to an initial blood sample obtained from the human at anytimeprior to determining the progression of the kidney disease. An“additional blood sample” refers to a blood sample obtained from thehuman at anytime after the first blood sample.

The protein content in an additional urine sample can be compared to theprotein content in the first urine sample and the creatinine content inthe additional blood sample can be compared to the first blood sample.An elevation in protein content in the additional urine sample aboveprotein content in the first urine sample and/or an elevation increatinine content in the additional blood sample above the creatininecontent in the first blood sample can be indicative of the progressionof the kidney disease in the human. Likewise, a decrease in proteincontent in the additional urine sample below the protein content in thefirst urine sample and/or an decrease in creatinine content in theadditional blood sample below the creatinine content in the first bloodsample is indicative that the kidney disease is not progressing in thehuman. Similarly, an essentially constant protein content in theadditional urine sample can indicate that the kidney disease is notprogressing.

Additionally the method of determining the progression of kidney diseasein the human can further include comparing blood urea nitrogen inadditional blood sample compared to a first blood sample. An elevationin blood urea nitrogen content in the additional blood sample aboveblood urea nitrogen content in the first blood sample is indicative ofthe progression of the kidney disease in the human. Similarly, adecrease in blood urea nitrogen content or an essentially constant bloodurea nitrogen content in the additional blood sample below blood ureanitrogen content in the first blood sample is indicative the kidneydisease is not progressing.

In another embodiment of the invention, the effectiveness of treatmentwith an iron chelator in a human suffering from a kidney disease isevaluated. Catalytic iron content in at least two urine samples areobtained from the human and compared. “Evaluating the effectiveness”refers to assessing the success of treating the human afflicted with akidney disease, in particular a progressive kidney disease, andespecially a progressive glomerular kidney disease with an iron chelatorto the extent that the kidney disease is treated (e.g., that the kidneydisease does not progress).

The urine samples obtained from the human to evaluate the effectivenessof treatment with the iron chelator includes a pretreatment urine sampleobtained from the human at a pretreatment time and at least onesubsequent urine sample obtained from the human at least at onesubsequent time after the pretreatment sample. A “pretreatment time”refers to anytime prior to the administration of the iron chelator. A“subsequent time” refers to anytime after the administration of the ironchelator.

A decrease in catalytic iron content in the subsequent urine sample toan amount below a catalytic iron content in the pretreatment urinesample is indicative of the effectiveness of treatment of the kidneydisease. Evaluation of the effectiveness of treatment with an ironchelator can further include comparing protein content in thepretreatment urine sample and at least one subsequent urine sample. Adecrease in protein content in the subsequent urine sample below proteincontent in the pretreatment urine sample is indicative of theeffectiveness of treatment with an iron chelator in a human sufferingfrom a kidney disease.

In yet another embodiment of the invention, evaluation of theeffectiveness of treatment with an iron chelator can further includemeasuring blood urea nitrogen content and creatinine content in a firstblood sample and in a subsequent blood sample obtained from the human.The blood urea nitrogen content and creatinine content in the subsequentblood sample is compared with blood urea nitrogen content and creatininecontent in the first blood sample. A decrease in blood urea nitrogencontent or creatinine content in the subsequent blood sample below bloodurea nitrogen content or creatinine content in the first blood sample isindicative of the effectiveness of treatment with an iron chelator in ahuman suffering from a kidney disease.

Thus, the effectiveness of treatment with an iron chelator can beevaluated by assessing decreases in urinary catalytic content, urinaryprotein content, blood urea nitrogen content and creatinine content in ablood sample compared to a pretreatment sample or a control sample.

Another embodiment of the invention relates to a method of slowing theprogression of a kidney disease in a human having catalytic iron inurine greater than about 15 nmol/mg of a reference protein, comprisingadministering an iron chelator to the human. The iron chelator candecrease the catalytic iron content in urine obtained from the urinethereby slowing the progression of the kidney disease in the human.

“Slowing the progression” of a kidney disease refers to reducing therate or speed at which, or degree to which a kidney disease advances ordevelops. For example, the progression of a kidney disease can be slowedso that additional glomerular damage or damage to the tubules in akidney that began with glomerular damage does not occur until a timelater than similar damage to the kidney in a human afflicted with thekidney disease who is not administered an iron chelator. Thus, slowingthe progression of kidney disease also refers to an increase in theamount of time (e.g., days, months, years) it would take to lead toprogressive kidney disease and, particularly progressive glomerularkidney disease or, ultimately, end-stage renal disease in a human whowas not administered the iron chelator.

A decrease in catalytic iron content after administration of the ironchelator to the human below a catalytic iron content measured beforeadministering the iron chelator can be used as an indice to assesswhether the progression of the kidney disease has been slowed.Additionally, or alternatively, other parameters which is indicative ofa slowing of the progression of kidney in a human administered an ironchelator can include, for example, decrease in the urinary proteincontent, blood urea nitrogen or creatinine in a blood sample obtainedfrom the human during or after administration of the iron chelatorcompared to urinary protein content, blood urea nitrogen or creatininein a blood sample obtained from the human before administering the ironchelator, or to a level about that of a control sample.

In yet another embodiment, the invention relates to a method ofidentifying a human suffering from a progressive kidney disease who willbenefit from treatment of the progressive kidney disease with an ironchelator. The phrase “who will benefit” refers to a human who has acatalytic iron content in urine greater than about 15 nmol/mg of areference protein or who can be treated with an iron chelator to, slowthe progression of the kidney disease, reduce the severity of the kidneydisease or halt the progression of the kidney disease in the human, andparticularly, progressive glomerular kidney disease in the human.

To identify a human who will benefit from treatment of a progressivekidney disease, catalytic iron content is measured in a urine sampleobtained from the human. A catalytic iron content in the urine samplegreater than about 15 nmol/mg of a reference protein identifies a humansuffering from a progressive kidney disease who will, or may, benefitfrom treatment of the progressive kidney disease with the iron chelator.

In still another embodiment, the invention relates to a method ofhalting the progression of a kidney disease in a human. “Halting” whenreferring to the progression of a kidney disease in a human refers tostopping, either temporarily or permanently, the progression of thekidney disease in the human. The progression of the kidney disease canbe halted, for example, by preventing or diminishing any further damageto any segment of the kidney (e.g., glomerulus, tubules, interstitum)damaged in the human with the progressive kidney disease. Thus, haltingof the progression of a kidney disease in a human can be a diminution inglomerular damage or prevention of damage to a segment of the kidney nototherwise damaged in the human with the progressive kidney disease(e.g., preventing tubular damage in a human with glomerular kidneydamage).

To halt the progression of a kidney disease in a human, catalytic ironcontent and protein content is measured in a pretreatment urine sampleobtained from the human. An iron chelator is administered to a humanhaving a catalytic iron content in the pretreatment urine sample in anamount greater than about 15 nmol/mg of a reference protein. The ironchelator can be administered in a dose of about 20 mg/kg body weight toabout 150 mg/kg body weight of the human. In a preferred embodiment, theiron chelator is deferiprone.

The catalytic iron content and protein content in the human is measuredin at least one subsequent urine sample obtained from the human duringor after the administration of the iron chelator. The catalytic ironcontent and protein content in the subsequent urine sample of the humanis compared with the catalytic iron content and protein content in aurine sample (e.g., pretreatment urine sample, first urine sample). Inone embodiment, the dose of iron chelator administered to the human isadjusted (e.g., increased or decreased compared to the dose initiallyadministered to the human) until the catalytic iron content in asubsequent urine sample of the human is below the control (e.g., 15nmol/mg of a reference protein). When the protein content in asubsequent urine sample is about twenty five percent (25%) below theprotein content in a control sample, the human is maintained on a doseof iron chelator until no further reduction in urinary protein occurs oruntil the urinary protein is essentially constant (e.g., for 3-4months). The dose of iron chelator is then reduced by a dose of about 10mg/kg body weight.

Urinary catalytic iron content is measured in at least one subsequenturine sample following the 10 mg/kg reduction in dose of the ironchelator. The dose of iron chelator can be further reduced by anadditional increment for 10 mg/kg body weight as long as the catalyticiron remains essentially constant or at a control level. Urinarycatalytic iron content, urinary protein, blood urea nitrogen andcreatinine in a blood sample are monitored following each reduction inthe dose of iron chelator. If the urinary catalytic iron, total urinaryprotein, blood urea nitrogen or creatinine in a blood sample increases,the dose of iron chelator can be increased by an increment of 10 mg/kgof body weight of the human. In a preferred embodiment, about everythree months the dose of iron chelator is reduced by about 10 mg/kg ofbody weight. The human can be maintained on a dose of iron chelatorabout 10 mg/kg body weight higher than the last dose which results in anessentially constant amount of urinary catalytic iron, total urinaryprotein, blood urea nitrogen or serum creatinine (also referred toherein as a “maintenance dose” of iron chelator).

Catalytic iron content in urine, urinary protein content, blood ureanitrogen or creatinine in blood can be measured while the human is on amaintenance dose of iron chelator and the dose of iron chelator adjustedto, for example, achieve a urinary protein content about that of acontrol sample. Similarly, if the catalytic iron content in a subsequenturine sample obtained from a human on a maintenance dose of ironchelator is greater than about 15 nmol/mg of a reference protein, thedose of iron chelator can be increased (e.g., to about 20-150 mg/kg ofbody weight).

The dose of iron chelator can be further increased if the catalytic ironcontent in the urine sample obtained from the human continues to risefollowing an adjustment in the dose of iron chelator above themaintenance dose. Similarly, the dose of iron chelator can be decreasedif the catalytic iron content in the urine sample obtained from thehuman decreases following an increase in the dose of iron chelatorcompared to the maintenance dose. The need to further adjust the dose ofiron chelator can be determined by monitoring catalytic iron content inurine, protein content in urine, blood urea nitrogen, creatinine in ablood sample, or any combination thereof.

The invention further relates to the use of iron chelators to diagnoseand treat humans exhibiting microalbuminuria. In a preferred embodiment,the iron chelator is deferiprone.

The term “microalbuminuria” refers to any disease, disorder, ailment orstate of health where urinary albumin is excreted at a rate of about20-200 μg/minute or about 30-300 mg/24 hours. (see, for example, Abbott,K. C., et al., Arch. Internal Med. 154:146-153 (1994), the teachings ofwhich are incorporated herein by reference in their entirety). Methodsto detect and diagnose microalbuminuria are well known to one of skillin the art and include radioimmunoassays, immunoassays with latexbodies, fluoroimmunoassays, enzyme immunoassays, agglutinationinhibition, immunoturbidimetry, immunonephelometry and radialimmunodiffusion assays. (Keen, H. et al., Lancet 2:913-916 (1968);Silver, A. et al., Clin. Chem 32: 1303-1306 (1986); Close, C. et al.,Diabet. Med. 4:491-492 (1987); Harmoinen, A. et al., Clin. Chim. Acta166:85-89 (1987); Marre, M. et al., Clin. Chem. 33:209-213 (1987);McCormik, C. P. et al., Ann. Clin. Lab Sci. 19:944-951 (1989); Cambiaso,C. L. et al., Clin. Chem. 34:416-418 (1988); Niwa, T. et al., Clin.Chim. Acta 186:391-396 (1990), the teachings of all of which areincorporated herein in their entirety). The levels of albumin or totalprotein in urine before and after treatment with the iron chelator canbe determined and the frequency or amount of doses required to alleviatemicroalbuminuria can be adjusted as needed for each individualundergoing treatment. Since microalbuminuria is associated with diabeticnephropathy in diabetic individuals (Mattock, M. B., et al., Diabetes41:736-741 (1992); Neil, A., et al., Diabetes Care 16:996-1003 (1993);Abbott, K. C., et al, Arch. Intern. Med. 154:146-153 (1994), theteachings of all of which are incorporated herein in their entirety) itis expected that the methods of the invention can be employed to haltthe progression of microalbuminuria and, thus, prevent development ofdiabetic nephropathy.

The amount effective to alleviate microalbuminuria can be an amount ofthe iron chelator that restores the levels of albumin in urine orprevents a further increase in albumin levels in the urine. In apreferred embodiment, the levels of albumin in urine are restored tolevels of about that in a control individual. The term “restore” refersto returning the albumin levels in urine to the levels observed beforethe individual developed microalbuminuria or to levels of about thatobserved in a control individual. The administration of iron chelatorsto humans afflicted with microalbuminuria can be used to halt theprogression of the microalbuminuria. In one embodiment, progression ofmicoralbuminuria is halted by administering to the human an ironchelator (e.g., about 20 mg/kg body weight to about 150 mg/kg bodyweight).

In another embodiment, the invention relates to a method of halting theprogression of microalbuminuria in a human, comprising measuringcatalytic iron content in a pretreatment urine sample obtained from thehuman and administering a dose of an iron chelator to a human having acatalytic iron content in the pretreatment urine sample above acatalytic iron content in a control sample. The catalytic iron contentin at least one subsequent urine sample obtained from the human ismeasured during or after the administration of the iron chelator andcompared with the catalytic iron content in the pretreatment urinesample, the control sample, or both. The dose of the iron chelatoradministered to the human is adjusted until the catalytic iron contentin the urine of the human is about that of the control sample.

A “control” level, when referring to the treatment of humans exhibitingmicroalbuminuria, is defined as described above for humans afflictedwith kidney diseases except the control level refers to a level ofurinary albumin, rather than a level of total protein as described inhumans afflicted with kidney disease.

Microalbuminuria can progress and ultimately lead to diabeticnephropathy, a progressive kidney disease (Mattock, M. B., et al.,Diabetes 41:736-741 (1992); Neil, A., et al., Diabetes Care 16:996-1003(1993); Abbott, K. C., et al., Arch. Intern. Med. 154:146-153 (1994),the teachings of all of which are incorporated herein in theirentirety). Thus, another aspect of the invention relates to a method ofhalting the progression of microalbuminuria in a human comprisingmeasuring catalytic iron content in a pretreatment urine sample obtainedfrom the human and administering a dose of deferiprone to a human havinga catalytic iron content in the pretreatment urine sample in an amountgreater than about 15 nmol/mg of a reference protein in a dose of about20 mg/kg body weight to about 150 mg/kg body weight of the human. Thecatalytic iron content is measured in at least one subsequent urinesample obtained from the human during or after the administration ofdeferiprone. The catalytic iron content in the subsequent urine sampleof the human is compared with the catalytic iron content in thepretreatment urine sample and the dose of deferiprone administered tothe human adjusted until the catalytic iron content in a subsequenturine sample of the human is below about 15 nmol/mg of a referenceprotein.

The methods of the present invention can be accomplished by theadministration of the iron chelator iron by enteral or parenteral means.Specifically, the route of administration is by oral ingestion (e.g.,tablet, capsule form). Other routes of administration as alsoencompassed by the present invention including intramuscular,intravenous, intraarterial, intraperitoneal, or subcutaneous routes, andnasal administration. Suppositories or transdermal patches can also beemployed.

The iron chelators can be used alone or in any combination whenadministered to the humans. For example, deferiprone can becoadministered with another iron chelator such as deferoxiamine to treata kidney disease (e.g., diabetic nephropathy, primaryglomerulonephritis, secondary glomerulonephritis, microalbuminuria). Itis also envisioned that one or more iron chelators can be coadministeredwith other therapeutics (e.g., steroids) to, for example, treat kidneydiseases, halt the progression of a kidney disease, reduce the severityof a kidney disease Coadministration is meant to include simultaneous orsequential administration of two or more iron chelators. It is alsoenvisioned that multiple routes of administration (e.g., intramuscular,oral, transdermal) can be used to administer one or more iron chelators.

The iron chelators can be administered alone or as admixtures withconventional excipients, for example, pharmaceutically, orphysiologically, acceptable organic, or inorganic carrier substancessuitable for enteral or parenteral application which do notdeleteriously react with the iron chelator. Suitable pharmaceuticallyacceptable carriers include water, salt solutions (such as Ringer'ssolution), alcohols, oils, gelatins and carbohydrates such as lactose,amylose or starch, fatty acid esters, hydroxymethycellulose, andpolyvinyl pyrolidine. Such preparations can be sterilized and, ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the iron chelator.

When parenteral application is needed or desired, particularly suitableadmixtures for the iron chelator are injectable, sterile solutions,preferably oily or aqueous solutions, as well as suspensions, emulsions,or implants, including suppositories. In particular, carriers forparenteral administration include aqueous solutions of dextrose, saline,pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,polyoxyethylene-block polymers, and the like. Ampules are convenientunit dosages. The iron chelators can also be administered viatransdermal pumps or patches. Pharmaceutical admixtures suitable for usein the present invention are well-known to those of skill in the art andare described, for example, in Pharmaceutical Sciences (17th Ed., MackPub. Co., Easton, Pa.) and WO 96/05309 the teachings of both of whichare hereby incorporated by reference.

The dosage and frequency (single or multiple doses) of iron chelatorsadministered to a human can vary depending upon a variety of factors,including the size, age, sex, health, body weight, body mass index, anddiet of the human; nature and extent of symptoms of the kidney diseasebeing treated (e.g., diabetic nephropathy, microalbuminuria), kind ofconcurrent treatment (e.g., steroids), complications from the kidneydisease, microalbuminuria, or other health-related problems. In apreferred embodiment, humans with a kidney disease are treated threetimes a day with a dose of iron chelator (e.g., deferiprone in 500 mgcapsules) at about 30 mg/kg to about 75 mg/kg body weight per day forabout 2-6 months. Other therapeutic regimens or agents can be used inconjunction with the iron chelator treatment methods of the presentinvention. For example, the administration of the iron chelator can beaccompanied by steroid administration. Adjustment and manipulation ofestablished dosages (e.g., frequency and duration) are well within theability of those skilled in the art.

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way.

EXEMPLIFICATION EXAMPLE 1 Urinary Catalytic Iron in Patients with KidneyDisease

Selection of Patients with Kidney Disease

Human patients (n=70) were used in these studies. Controls were healthyhuman patients with no history of kidney disease, hypertension, diabetesor other diseases that affect the kidney. Males and females were used inthe studies. Ages of the human patients ranged from 4 years of age to 70years of age. Ethnicity of the patients included Caucasians,African-American and Hispanics.

Kidney disease was detected in patients by the presence of an abnormalamount of protein in the urine and/or an abnormal renal function. Anabnormal amount of protein in urine is an amount of total proteingreater than about 0.15 g/24 h measured by an adaptation of pyrogallolred-molydate method described by Fujita, Y., et al., Bunseki Kgaku32:379-386 (1983), the teachings of which are hereby incorporated byreference in their entirety. Many patients, as part of their routinework up, had received kidney biopsies providing diagnosis of the natureof the kidney disease.

Glomerular filtration rate was assessed in humans by measuring serumcreatinine (normal values 0.96 mg/dl for women, and 1.16 mg/dl formen—values above 1.4 mg/dl considered abnormal), creatinine clearance orboth. Creatinine clearance was calculated by the standard formula Cr=UcrV/PCr where Cr=clearance of creatinine (ml/min); Ucr=urine creatinine(mg/dl), V=volume of urine, and PCr=plasma creatinine (mg/dl).

Patients with kidney disease included the following groups of patients:

Glomerulonephritis. This diagnosis was based on patients with abnormallyhigh amount of urinary protein (greater than about 150 mg/24 hours)including patients with nephrotic syndrome (greater than about 3.5 gmprotein). The diagnosis of glomerulonephritis was made on histologicalexamination of the kidney, which included light microscopy,immunofluorescence, and electron microscopy using routine methods.Diagnoses included both primary causes such as membranous nephropathy,focal segmental sclerosis, IgA nephropathy, membranoproliferativeglomerulonephritis, and crescentic glomerulonephritis as well assecondary causes such as, systemic lupus erythematosis, hemolytic uremicsyndrome or Henoch-Schonlein purpura.

Microalbuminuria Patients with diabetes who had microalbuminuria hadurinary albumin in a range between about 30 mg to about 300 mg for 24hours as measured by a method which uses the rate of increase in lightscattered from particles suspended in solution as a result of complexesformed during an antigen-antibody reaction or by the dipstick method.

Diabetic Nephropathy. Patients with diabetes who had diabeticnephropathy with overt proteinuria (greater than about 150 mg/24 hours)in 24-hour sample. The diagnosis of diabetic nephropathy was based onpresence of overt proteinuria, history of diabetes, and presence ofretinopathy on clinical examination.

Ischemic nephropathy was based on clinical and radiological findings.

A urine sample was obtained from humans with and without (control)kidney disease and assessed for catalytic iron content and creatininecontent using established methods as described below.

Urinary Catalytic Iron and Creatinine in Humans With and Without KidneyDisease

Catalytic iron content was measured in urine using a well establishedbleomycin assay (Gutteridge J. M. C., et al., Biochem J 199:263-265(1981), the teachings of which are incorporated herein in theirentirety).

In vivo iron is primarily bound to heme or non-heme proteins and doesnot directly catalyze the generation of hydroxyl radicals or a similaroxidant (Gutteridge J. M. C., et al., Biochem J 199:263-265 (1981), theteachings of which are incorporated herein in their entirety). In thecatalytic iron assay used in these studies, the antibiotic bleomycindetects iron complexes capable of catalyzing free radical reactions.Bleomycin, in the presence of an iron salt and a suitable reducingagent, binds to and degrades DNA with the formation of a product thatreacts with thiobarbituric acid to form a chromogen. The binding of thebleomycin-iron complex to the DNA makes the reaction site specific andantioxidants rarely interfere. Thus, the iron detected by this methodwas a measure of iron available from the sample to bleomycin. The assayconditions prevent interference from iron containing proteins. Ironbound to transferrin, lactoferrin, ferritin or iron containing enzymesis not detected in the bleomycin assay (Gutteridge J. M. C., et al.,Biochem J. 199:263-265 (1981), the teachings of which are incorporatedherein in their entirety).

Specifically, the assay was performed as follows:

Reagent preparation

-   -   1. Chelex-treated pyrogen free H₂O (Chelex H₂O)        -   Add 5 g chelex 100 resin (Bio-Rad, Cat#: 142-2842: 200-400            mesh, sodium form) to 100 ml pyrogen free water (Baxter) and            shake well. Stand overnight at 4° C. and filter off the            Chelex with bottle filter system (0.22 μm filter). After            Chelex treatment of all solutions used in the assay, the            Chelex must be carefully removed to avoid contamination by            resin.    -   2. DNA (1 mg/ml)        -   Add 50 mg DNA (Type I: calf thymus, Sigma D-1501) to 50 ml            Chelex H₂O and allow to stand at 4° C. overnight to effect            solution. Treat this solution with 15 mg Chelex 100 resin            overnight at 4° C. and centrifuge at 3000 rpm for 30 min to            remove Chelex. If there are still some Chelex particles in            the solution, centrifuge it again at 3000 rpm about 15 min.            The reconstituted DNA is suitable for use in the assay for            about 7 days after mixing when stored at 4° C.    -   3. Bleomycin sulfate (1 U/ml)        -   15 U bleomycin sulfate (Bleoxane, Nippon Kayaku Co. Ltd,            Tokyo, Japan) in 15 ml Chelex H₂O. The reconstituted            bleomycin is suitable for use in the assay for about 1 month            after preparation.    -   4. Magnesium chloride (50 mM)        -   Add 0.1 g MgCl₂.6H₂O (FW. 203.3) in 10 ml Chelex H₂O. Treat            the solution with 3 g Chelex 100 resin and centrifuge at            3000 rpm for 10 min to remove Chelex.    -   5. Hydrochloric acid (40 mM)        -   33.2 μl HCl (Ultrapure, Baker 6900-05) in 10 ml Chelex H₂O.    -   6. NaOH (40 mM)        -   16 mg NaOH in 10 ml Chelex H₂O.    -   7. Ascorbic acid (8 mM)        -   70 mg ascorbic acid (FW. 176.1) in 1 ml Chelex H2O. Treat            the solution with 50 mg Chelex 100 resin and centrifuge at            3000 rpm for 30 min to remove Chelex. Dilute 1:50 with            Chelex H2O before use. Prepare immediately prior to use in            the assay.    -   8. EDTA (0.1 M)        -   0.37 g EDTA in 10 ml Chelex H₂O.    -   9. Thiobarbituric acid (TBA) (1% w/v in 50 mM NaOH)        -   1 g TBA in 100 ml of 50 mM NaOH (NaOH prepared in Chelex            H₂O).    -   10. HCl (25% v/v)        -   25 ml HCl (Ultrapure, Baker Catalog #6900-05) in 100 ml            Chelex H₂O.    -   11. Standard Iron        -   16 mg FeCl₃ (FW. 162.2) in 10 ml Chelex H₂O. Dilute 1:100            for stock solution (100 nmol/ml), then dilute 1:1 to make            the standard concentrations as 50. 25, 12.5, 6.25, and 3.125            nmol/ml and Chelex H₂O. Prepare standards immediately prior            to use in the assay.

Procedure

-   -   1. To remove particulates, urine samples were centrifuged at        about 3000 rpm for about 10 min and the resulting supernatant        used in the bleomycin assay.    -   2. Place the following in a plastic tube in the following order:        -   0.5 ml DNA        -   0.05 ml bleomycin sulfate (for blanks add Chelex H₂O)        -   0.1 ml MgCl₂        -   0.1 ml standard Fe solution or urine samples        -   0.1 ml ascorbic acid    -   3. Mix well before and after the addition of ascorbic acid. For        each sample or standard prepare a parallel blank. The sample and        standard blanks are identical to their test reaction mixtures        except that bleomycin is omitted    -   4. Adjust pH of all standards, samples and their blanks to about        pH 7.2-7.8 using 40 mM HCl or 40 mM NaOH.    -   5. Incubate all tubes in temperature controlled shaker at 100        rpm, about 37° C. for about 2 h.    -   6. Stop the reaction by addition of 0.1 ml EDTA.    -   7. Add 1 ml TBA and 1 ml 25% HCl and mix well.    -   8. Transfer the mixture into new or acid-washed glass tube and        heat at about 100° C. for about 15 min.    -   9. Transfer the solution in to 4.5 ml cuvette after cooling and        read absorbency at 532 nm against the corresponding blank        (without bleomycin).    -   10. Calculate the amounts of bleomycin-detectable iron content        in test samples from the standard curve obtained in each        experiment. The absorbency of the zero standard (contains only        Chelex H₂O) reflecting iron contamination in the reagents is        subtracted from all test samples.    -   11. Urine bleomycin-detectable iron is expressed as nmol/mg        creatinine. Creatinine in the urine sample is measured using a        creatinine assay kit (Sigma Chemical Co., St. Louis, Mo. 555-A).        Statistical Analysis

Data were expressed as the mean ±SEM of nmoles catalytic iron/mgcreatinine. Significant differences between group means was determinedusing an unpaired t-test. The patient data was compared to controlsusing a paired t-test and p-value of <0.05 was considered significant.

Results and Conclusions

As shown in FIG. 1, Table 1 and Table 2, urinary catalytic iron waselevated in patients with a wide variety of kidney diseases compared topatients without kidney disease. Thus, urinary catalytic iron plays arole in kidney disease and is indicative of a kidney disease in a human.

EXAMPLE 2 Treatment of Patients with Kidney Disease with an IronChelator

Selection of Patients with Kidney Disease

The diagnosis of the kidney disease was made by performing a kidneybiopsy and included patients with membranoproliferativeglomerulonephritis, membranous nephropathy, focal segmental sclerosis,IgA nephropathy, and diabetes as described above.

Iron Chelator Treatment of Patients with Kidney Disease

Patients with kidney disease who had abnormally high urinary proteinwere treated with the iron chelator deferiprone(1,2-dimethyl-3-hydroxypyrid-4-one) (L1). Deferiprone was synthesized aspreviously described capsule (Kontoghiorghes A. J., et al., InorganicaChimica Acta 136:L11-L12 (1987), the teachings of which are herebyincorporated by reference in it entirety) and formulated into capsulescontaining 500 mg of deferiprone per capsule.

Urinary protein and serum creatinine was measured as described abovebefore the administration of deferiprone. Patients were treated by oralingestion of deferiprone in a range between about 30 mg and about 75 mgdeferiprone per kilogram body weight per day 3 times a day. Urinaryprotein and serum creatinine was obtained 2-6 months after theadministration of the iron chelator. As shown in Table 3, treatment withthe iron chelator significantly decreased the amount of total urinaryprotein and serum creatinine in patients with kidney disease. Totalurinary protein and serum creatinine measured before (e.g.,pretreatment) and after the administration of deferiprone were comparedusing a paired t-test and p-value of <0.05 was considered significant.

These data show that the iron chelator treated the kidney diseases.Therefore, it is believed that the effect of catalytic iron can besignificantly reduced or prevented from acting directly or indirectly onthe kidney to injure the kidney, thereby causing kidney disease, whichif left untreated (no iron chelator) would result in an increase intotal urinary protein content and serum creatinine.

TABLE 1 Urinary Catalytic Iron In Patients with Progressive KidneyDisease Catalytic Iron 24 hr prot Serum Cr CrCl (Nmol/mg Age/Sex/RaceDiagnosis (mg/24 hrs) (mg/dl) (ml/min) creatinine) 20 F W Membranous8900 1.1 120.0 33.4 23 M W Membranous 4200 2.3 22.3 50 F B SLE 5000 1.957.0 90.7 51 F B SLE 5000 1.4  9.1 64 M W SLE 3700 0.9 11.0 45 F W SLE3700 0.6 69.0 38 F B SLE 6843 4.4 70.0 38 F B FSGS 5000 2.6 52.7  7 M WFSGS 20790 2.1 30.6 48.6 15 F B FSGS 2360 0.8 110.2 43.7 14 F B FSGS16350 2.3 38.6 17.3 32 M B FSGS 300 2.2 20.1 40 F B FSGS 5200 1.7 61.640 M W FSGS 2.1 28.0 40 M B FSGS 15730 3.3 20.0 118.0  11 F W HSP 111200.6 139.3 26.1 <1 M W HUS 0.7 84.2 16 M B IgA 830 1.6 58.4 19.1 <1 M BMPGN 2104 0.5 75.0 66.8 54 M MPGN 3.8 43.0 40 M W MPGN 16260 106.0  65 FW Creacentic 5.9  5.4 Mean 48  SEM 7 Number 22  P    <0.0001 IschemicNephropathy 45 F W Isch Neph 1.8 92.0 80 F W Isch Neph 2.0 96.0 Mean94.0 SEM  2.0 Number 2 P   <0.01 SLE: Systemic Lupus Erythematosus; FSGS= Focal Segmental Glomerulosclerosis; HSP = Henoch-Schonlein Purpura;HUS = Hemolytic Uremic Syndrome; MPGN = MembranoproliferativeGlomerulonephritis; Isch Neph = Ischemic Nephropathy SLE, HSP, HUS andIschemic Nephropathy are secondary causes of renal disease. P comparedto control value of 8.1 ± 1.4, n = 23, using unpaired t test Isch Neph =Ischemic Nephropathy prot = protein Cr = creatinine CrCl = creatinineClearance

TABLE 2 Urinary Catalytic Iron In Patients with Diabetic NephropathyMicroalbuminuria 24 hr prot Serum Cr CrCl Catalytic Iron Age/Sex/Race(mg/24 hrs) (mg/dl) (ml/min) (nmol/mg creatinine) 69 F W 0.8 59.3 77 M W1.4 84.1 56 F W 0.7 75.9 39 F B 0.6 10.2 47 F W 1.0 46.3 50 M B 1.3103.0 23.0 56 M W 2.4 46.0 21.0 61 M W 1.2 90.0 36.0 69 M W 1.1 77.025.0 73 M B 1.4 54.0 58.0 78 M W 1.1 71.0 82.0 59 F B 1.2 116.7 44.0 30M B 1.4 68.0 86.0 Mean 50.0 SEM  7.0 Number 13  P    <0.0001 DiabeticPatients with Overt Proteinerria 61 M B 4800.0 5.3 56.2 56 M B 8000.03.0 20.0 67 F B 3816.0 3.1 25.0 42.0 42 F W 8300.0 7.2 10.0 32.6 51 F B11200.0 2.8 31.0 53.8 32 M W 6899.0 6.8 44.5 61 M B 11363.0 5.4 30.2 73M W 1064.0 3.0 27.3 71 F B 3200.0 1.6 49.0 79 F W 1350.0 3.0 25.0 Mean38.0 SEM  4.0 Number 10  P    <0.0001 P compared to control value of 8.1± 1.4, n = 23, using unpaired t test Microalbuminuria is defined asurinary albumin between 30 to 300 mg/24 hrs as measured quantitavely ina 24 hour sample or by a dipstick in a spot urine. prot = protein Cr =creatinine CrCl = creatinine Clearance

TABLE 3 Urinary protein and creatinine levels in patients with kidneydisease before and after treatment with an iron chelator 24-Hr UrinaryProtein Wt grams/24 hrs Reduction Creatinine mg/dl Pt. # Sex Age (kg)Diagnosis* Before After Grams % Before After 1 F 52 60 MPGN 2.6 1.2 1.454 1.3 0.8 2 5 F 40 73 Membranous 2.8 0.6 2.2 78 1.4 0.9 3 F 29 42Chronic GN 3.4 1.5 1.9 49 1.0 0.9 4 M 33 74 Membranous 1.2 0.2 1 83 1.41.3 5 F 40 66 Membranous 6.5 4 2.5 38 0.8 0.9 6 M 28 FSGS 2.8 1.6 0.7 257 10 F 18 36 FSGS 4.2 1.6 2.6 62 0.7 0.6 8 M 14 37 Mesangial 6 0.05 5.9599 0.6 0.5 proliferation/ IgA 9 M 3 14 DPGN 5.8 2.8 2 34 10 F 12 MPGN4.6 2.5 2.1 44 0.4 0.5 11 M 32 77 Diabetes 0.538 0.1 0.43 80 1.6 1.4 1215 M 45 Mesangial 4.5 3.8 0.7 15 proliferation/ IgA 13 M 35 50 Membrane5.3 4.8 0.5 9 0.8 0.7 14 M 32 62 Membranous FSGS 4.5 4.6 0.1 2 2.1 1.5Mean 3.91 2.13 1.70 48 1.10 0.90 SD 1.78 1.67 1.49 30 0.50 0.35 SEM 0.470.45 0.39 8 0.15 0.10 N 14 14 14 14 11 11 p < 0.001 p < 0.001 p < 0.001DPGN: Diffuse proliferative glomerulonephritis; MPGN:Membranoproliferative glomerulonephritis; FSGS: Focal segmentalglomerulosclerosisEquivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A method of treating progressive kidney disease in a human,comprising the step of administering an iron chelator to the human. 2.The method of claim 1, wherein the iron chelator is selected from thegroup consisting of deferiprone, deferoxamine, polyanionic amines andsubstituted polyaza compounds.
 3. The method of claim 1, wherein theiron chelator is administered in an amount that causes urinary catalyticiron content of the human to be essentially constant.
 4. The method ofclaim 1, wherein the iron chelator is administered at a dose in a rangeof between about 20 mg/kg body weight and about 150 mg/kg body weight ofthe human per day.
 5. The method of claim 1, wherein the human issuffering from a progressive kidney disease of at least one memberselected from the group consisting of primary glomerulonephritis andsecondary glomerulonephritis.
 6. The method of claim 1, wherein the ironchelator halts progression of the kidney disease.
 7. The method of claim1, wherein the iron chelator reduces the severity of the progressivekidney disease.
 8. The method of claim 1, wherein the iron chelator isadministered in multiple doses.
 9. The method of claim 1, wherein theiron chelator is administered orally.
 10. The method of claim 1, whereinadministration of the iron chelator reduces the rate of loss of renalfunction.
 11. The method of claim 10, wherein administration of the ironchelator reduces the rate of progression of proteinurea.
 12. The methodof claim 11, wherein administration of the iron chelator reduces therate of increase of urinary creatinine.
 13. The method of claim 10,wherein administration of the iron chelator reduces deterioration ofglomerular filtration rate.
 14. The method of claim 10, whereinadministration of the iron chelators reduces at least one member of thegroup consisting of protein in urine, blood urea nitrogen and serum orplasma creatinine.
 15. The method of claim 5, wherein the primaryglomerulonephritis is at least one member selected from the groupconsisting of membranous nephropathy, focal segmental sclerosis, IgAnephropathy, membranoproliferative glomerulonephritis and crescenticglomerulonephritis.
 16. The method of claim 5, wherein the secondaryglomerulonephritis is at least one member selected from the groupconsisting of diabetic nephropathy, systemic lupus erythematosis,hemolytic uremic syndrome and Henoch-Schonlein purpura.
 17. The methodof claim 1, wherein the progressive kidney disease is a ischemicnephropathy.
 18. The method of claim 1, wherein the progressive kidneydisease is consequent to damage to an interstitium of a kidney.